The Study On The Relationship Of FMDV Infection And Host EBP Gene In Single Cell

Foot-and-mouth disease virus(FMDV)belongs to the family Picornaviridae.It can cause acute infection as well as persistent infection.Under natural conditions,FMDV could not only cause acute infection in cloven-hoofed animals but also produce persistent infection(PI)in the low-toxicity form in animals.Under in vitro cell cultivation,acute infection by FMDV could cause the death of host cells via cytopathic effect(CPE).By changing the infection condition and the screening method of surviving cells after infection,we can obtain cell lines that were persistently infected by FMDV.The establishment of persistent infection cell lines will help us understand the relationship between the virus and host cells.Previous studies in our lab have shown that the expression of EBP(emopamil binding protein)gene was up-regulated in BHK-21 cells aftelr FMDV acute infection,whereas EBP expression was down-regulated in FMDV persistently infected cells.EBP encodes a sterol isomerase widespread in mammalian cells.The investigation on the changes of EBP gene expression during FMDV acute infection and persistent infection will help us understand the mechanisms by which acute infection and persistent infection are established as well as the roles of EBP gene in two infection models.In this study,318 single cells across four generations PI28,PI38,PI55 and PI68 were picked up from FMDV persistent infection cell lines.Transcriptomic studies in each single cell were performed to explore the trend of changes in EBP gene expression in BHK-21 cells persistently infected by FMDV,and thereby to reveal the mechanism of randomness of gene expression and the heterogeneitity of cells caused by viral infection.The results are listed as follows:1)In BHK-21 cells acutely infected by FMDV at 20000 pfu at 8 h.p.i,the expression level of EBP gene was upregulated to 1.36 times of the amount in normal cells.The percentage of single cells whose EBP expression level was upregulated was 11.9%among total cells,which is twice the number of cells at 4 h.p.i.The infection rate was 90%at 4 h.p.i and reached 100%at 8 h.p.i.The amount of virus increased by four orders of magnitude in just four hours from 4 h.p.i to 8 h.p.i.2)In FDMV-infected BHK-21 cells that overexpress EBP gene,the level of FMDV replication is 2.3 times the level of control cells,suggesting that upregulation of EBP expression can promote FMDV replication to some degree.3)The expression levels of 3D gene(FMDV gene)and host EBP gene were measured in 318 single cells that are persistently infected by FMDV.We found that the expression levels of viral 3D gene and host EBP gene changed dynamically at different subculture periods during viral persistence infection.4)In PI28,the proportion of single cells carrying virus was relatively low(only 66.7%).FMDV now was at the stage of transition from acute infection to persistent infection.The mean EBP expression in PI28 cells was the highest among all generation cells and was also higher than the mean in normal single BHK-21 cells.The proportion of cells with upregulated EBP expression was 62.9%.The highest EBP gene copy number was 982 copies/cell,which is 1.24 times the level of normal cells.5)In PI38,EBP gene expression gradually decreased.The percentage of cells with EBP expression level lower than that of normal cells was 78.2%.The proportion of single cells carrying virus increased(98.7%of the total).The expression level of viral 3D gene in single cells reached the peak among all generations.66.7%of cells showed the expression level of 3D gene higher than the mean of PI28.The highest 3D gene copy number was 17576 copies/cell,which is 3.8 times the mean of PI28.6)In PI55 and PI68,the proportions of single cells carrying virus were 100%and 95.8%,respectively,which is close to reach a balance.This suggests that persistent viral infection was stabilized.The expression level of host EBP gene declined rapidly,reached to a minimum in PI68 generation.The percentage of downregulated cells was 95.6%.The average expression level of EBP was only a quarter of that in normal BHK-21 cells.The expression level of FMDV 3D gene also decreased to the lowest in PI68.The percentage of downregulated cells was 91.2%compared with that of PI28 generation.None of cells showed upregulation of both EBP gene and 3D gene.In this study,we investigated the variation trend of the FMDV virus 3D gene and host EBP gene in different generations of persistently infected BHK-21 cells.Our results showed that this approach can reflect the real situation in each cell.Our method could detect the effect that may be hidden by homogenization of population data and could reveal the randomness of gene expression and the heterogeneitity of cells during viral infection.Our studies in single cells showed that the establishment of FMDV persistent infection in cells is a dynamic process.It is the result of mutual adaption between the virus and host cells and the result of co-evolution that eventually reaches a balance.